The invention relates to the extracellular matrix of cartilage, and more specifically to cartilage matrix protein (CMP) and link protein (LP).
Connective tissues, in general, have an abundant extracellular matrix, synthesized and maintained by specialized cells. The interactions among these cells, the uniquely combined extracellular matrix components, and the water, electrolytes, and proteins in the extracellular fluid of each type of connective tissue determine the tissue's functional characteristics.
In cartilage, one major component of the extracellular matrix is the collagen fibril. As the principal tensile element, the collagen fibril plays a key role in the structural stabilization of cartilaginous tissues. The fibril consists of a core of type XI collagen surrounded by type II collagen, and on the periphery there is type IX collagen covalently attached to the type II collagen (Mendler et al. J. Cell Biol. 108: 191-197, 1989).
Several non-collagenous proteins that interact with collagen fibrils, including decorin (Vogel et al., Coll. Rel. Res. 7: 104-114, 1987), cartilage matrix protein (CMP) (Paulsson et al., Biochem. J. 197: 367-375, 1981; Winterbottom et al., Dev. Dynamics 193: 266-276, 1992), fibromodulin (Hedbom et al., J. Biol. Chem. 264: 6898-6905, 1989) and collagen binding protein (Chandrasekhar et al., Proc. Natl. Acad. Sci. USA 83: 5126-5130, 1986 ), have also been described. The role of these proteins in the organization of the fibrils is not clear, although it has been recently reported that CMP binds to type II collagen (Winterbottom et al., supra).
CMP is a homotrimer of disulfide bond linked subunits, and contains a domain with significant homology to epidermal growth factor, and two homologous repeat sequences (CMP-1 and CMP-2) which have homology to regions within von Willebrand's factor, complement factor B, complement factor C2, Type VI collagen, and the .alpha. chains of the integrins Mac-1, p150,95 and LFA-1 (Kiss et al., J. Biol. Chem. 264: 8126, 1989; Argraves et al. Proc. Natl. Acad. Sci. USA 84: 464, 1987). These repeat sequences have been reported to contain the regions responsible for collagen binding (Winterbottom et al., supra).
Most of the extracellular space between the collagen fibrils is occupied by the second major component of the cartilage extracellular matrix, the ternary complex, which is composed of monomers of the large cartilage proteoglycan, aggrecan, link protein (LP), and hyaluronic acid (HA). The polyanionic glycosaminoglycan side chains of each aggrecan monomer are covalent modifications of the core protein and restrain large volumes of water and ions within the matrix, thus providing the shock-absorbing properties of the cartilage tissue (Hascall, J. Supramol. Struc. 7: 101-120, 1970). As many as 100 aggrecan monomers bind to a single HA polymer through amino acid residues contained in the amino-terminal globular domain. These interactions are stabilized by the binding of a single molecule of LP independently to both aggrecan and HA (Hascall, in Biology of Carbohydrates, Vol 1., pp. 1-49, ed. Ginsberg, C., Wiley, N.Y., 1981).
The amino acid sequence of LP has revealed that this protein is composed of an NH.sub.2 -terminal domain which possesses homology with immunoglobulin like proteins (Bonnet et al., Biochem. Biophys. Acta. 873: 152, 1986) and two tandemly repeated sequences that have homology with the HA-binding region of aggrecan (Deak et al. Proc. Acad. Sci. USA 83: 3766, 1986; Doege et al., Proc. Acad. Sci. USA 83; 3761, 1986; Neame et al., J. Biol. Chem. 261: 3519, 1986). Based on this sequence homology it has been suggested that these tandemly repeated sequences contain the sites for interaction with HA (Deak et al., supra; Neame et al., J. Biol. Chem. 260: 12402, 1985; Goetinck et al., J. Cell Biol. 105: 2403, 1987). In addition to being present in large amounts in cartilage, LP has also been found in a number of embryonic tissues including aorta, dorsal skin, kidney, and eyes, as well as in the connective tissue along the entire digestive tract (Gardell et al., BBRC 95: 1823, 1980; Stirpe et al., Dev. Biol. 137: 419, 1990; Poole et al., J. Cell Biol. 93: 910, 1982; Ripellino et al., J. Cell Biol. 108: 1899, 1989; Binette et al., Mol. Biol. Cell 3: 224, 1992). It has been proposed that the presence of LP in a wide variety of tissues indicates that this protein may also play a role in the stabilization of the extracellular matrix of non-cartilaginous tissues (Binnette et al., supra).
To date, the nature of the macromolecular interactions between the major components of the extracellular matrix of cartilage and non-cartilaginous tissues are unknown, but it has been recognized that this interaction is critical for the growth and maintenance of these tissues.